Identification of PDZ Interactions by Affinity Purification and Mass Spectrometry Analysis.

Résumé

Identification of protein networks becomes indispensable for determining the function of a given protein of interest. Some proteins harbor a PDZ binding motif (PDZBM) located at the carboxy-terminus end. This motif is necessary to recruit PDZ domain proteins which are involved in signaling, trafficking, and maintenance of cell architecture. In the present chapter, we present two complementary approaches (immunopurification and peptide-based purification procedures) followed by mass spectrometry analysis to identify PDZ domain proteins associated to a given protein of interest. As proof of example, we focus our attention on TANC1 which is a scaffold protein harboring a PDZBM at its carboxy-terminus. Using these two approaches, we identified several PDZ domain containing proteins. Some of them were found with both approaches, and some were specifically identified using peptide-based purification procedure. This exemplifies advantages and differences of both strategies to identify PDZ interactions.